Feline caliciviruses (FCV) were first described in 1957 (Fastier, 1957). Feline caliciviruses and feline herpesviruses are the two principal sources of viral diseases of the upper respiratory tract in cats. The FCV viruses affect a large number of animals, with FCV carrying infection rates of the order of 15 to 25%, and an anti-FCV seroprevalence of 70 to 100% (Coutts, 1994; Ellis, 1981; Harbour, 1991; Reubel, 1992). After an initial phase of hyperthermia, these respiratory diseases are generally accompanied by buccal ulcerations on the palate, tongue, lips, and/or nose, rhinitis, chronic stomatitis, conjunctivitis, and possibly anorexia and asthenia. The FCV viruses can also cause pneumonia, enteritis, and articular pain, also known as lameness syndrome.
In general, FCV can be isolated from 15 to 25% of the overall cat population (Harbour, 1991), 45 to 60% of cats with upper respiratory tract infection (Harbour, 1991; Reubel, 1992) and 50 to 92% of cats with chronic stomatitis (Knowles, 1989; Harbour, 1991).
The FCV virus is only transmitted horizontally; to date, there are no known cases of vertical transmission from the mother to its kitten during gestation (Johnson, 1984). Instead, FCV is transmitted by contact between infected animals and healthy animals or through exposure to droplets in the air as occurs through sneezing (Wardley, 1976).
Feline calicivirus of the Caliciviridae familly is a non-enveloped virus, comprising a single-stranded positive-sense RNA genome that is polyadenylated and is about 7.7 kilobases in size (Radford, 1997). The FCV capsid is comprised of a single major capsidal protein of 66 kDa (kilodalton), the p66 protein. A review of the molecular biology of the caliciviruses can be found in Clarke and Lambden.
Like many RNA viruses, a large heterogeneity exists within the viral population of FCV. The antigenic variations, demonstrated since the beginning of the 1970s by cross-serum neutralization experiments, make it possible to classify the FCVs into several viral strains or quasispecies (Radford, 1997).
Several FCV strains have been identified and isolated, in particular strain F9 (deposited with the American Type Culture Collection or ATCC under the accession number VR-782), strain 2280 (ATCC VR-2057), strain KCD (ATCC VR-651) and strain CFI (ATCC VR-654).
Vaccination against FCV was introduced at the end of the 1970s using attenuated FCV strains, mainly strain F9 which was isolated in the United States in 1958 (Bittle, 1960) or those strains derived from F9 by passage in vitro or in vivo (“F9-like”). These attenuated vaccines comprises the majority of the commercial FCV vaccines available.
Inactivated vaccines are also commercially available, and all of these inactivated vaccines contain an adjuvant. These inactivated vaccines mainly use strains 255 and 2280, which were isolated in the United States in 1970 in a cat with a pneumonia (Kahn and Gillepsie, 1970; Povey, 1980) and in 1983 in a cat suffering from lameness (Pedersen, 1983; Pedersen and Hawkins, 1995), respectively.
Inactivation of FCV for use in vaccines may be accomplished by a variety of methods, including the use of formalin. For example, Povey describes a formalin inactivated and adjuvanted FCV preparation used in kittens (Povey, 1978).
U.S. Pat. No. 6,534,066 describes the use of new strains of FCV for the production of FCV vaccines. When the vaccine is an inactivated one, the inactivation occurs through chemical means (e.g. formalin, or formaldehyde, β-propiolactone, ethylenimine, binary ethyleneimine) and/or a heat treatment. Preferably, the virus is inactivated by ethyleneimine. The vaccines are preferably adjuvanted, for example with an oil-in-water emulsion as described in example 8 of the patent.
U.S. Pat. No. 6,355,246 describes both attenuated and inactivated FCV vaccines that preferably comprise an adjuvant. In this patent, inactivation is accomplished through the use of formaldehyde or binary ethyleneimine (BEI).
As mentioned previously, the inactivated vaccines typically contain an adjuvant to improve the immune response and to induce a better protection against heterologous FCV strains emerging in the cat population. However, adjuvanted vaccines induce a higher rate of local adverse reactions than non-adjuvanted ones (Gobar, 2002) and thereby increase the risk of vaccine-associated fibrosarcomas at the injection site (Baker, 1998).
Non-adjuvanted FCV vaccines are typically modified live vaccines usually containing the F9 strains as described above. The residual virulence of FCV F9 has been incriminated by several authors in post-vaccinal calicivirosis (reversion to virulence) (Dawson, 1993). FCV modified live strains are implicated in the emergence of new antigenic variants in the field (Radford, 1997). Therefore, the safety of modified live vaccines is questionable.
Although only one FCV serotype exists, antigenic variation between FCV isolates is observed and new field isolates are regularly identified (Lauritzen, 1997).
Accordingly, because of antigenic drift over time, antisera produced against vaccine strains isolated in the 1960-70s, including strains such as F9, 255 or 2280, neutralize only a few isolates of those calicivirus strains prevalent in the 1990s and 2000s. For example, the anti-F9 serum neutralizes 43% of the American isolates of the period 1990-1996, compared to 56% of the isolates for the period 1980-89 and 86% of the isolates for the period 1958-79, and only 10% of the English isolates of the period 1990-96 (Lauritzen, 1997). Therefore, attenuated and inactivated vaccines from old FCV strains no longer offer sufficient protection against recent FCV strains.
Despite the use of vaccination against FCV since the end of the 1970s, FCV-associated diseases continues to be a significant clinical problem. And, as described previously, new hypervirulent strains have recently arisen. Several mechanisms explain the persistence of FCV infection and FCV-related diseases in face of vaccination, including the lack of broad cross protection afforded by vaccinal strains due to the evolution of the FCV population under the immune pressure induced by vaccination (Geissler, 1997); vaccinal strains from attenuated vaccines may contribute to acute and chronic FCV infection (Dawson, 1993; Pedersen, 1995; Radford, 1997); both inactivated and live vaccines protect the cat against clinical disease but not against infection (Pedersen, 1995); and, FCV is able to evolve and escape from immune pressure by giving rise to mutants which are more vaccine resistant (Knowles, 1990; Johnson, 1992).
As a result, the current calicivirus vaccines must be replaced by vaccines that are more adapted to the current epidemiological situation and which provide greater cross-neutralization against the isolates currently identified in the feline populations. A promising vaccine would be one that is either inactivated or recombinant and which is based on a newer strain of FCV. Recently, outbreaks of a very severe calicivirosis have been noted in the United States and other countries. One of these hypervirulent and immunodominant strains has been selected as vaccine candidate and is described herein as an alternative to the traditional FCV vaccines.
Furthermore, in cats an additional problem arising from FCV vaccination is the presence of inflammation at the injection site, often as the result of the presence of an adjuvant in the vaccine, which may be a factor in post-vaccinal fibrosarcomas. Therefore, local tolerance of the vaccine is of strategic importance, and should be considered when developing new vaccines such that the ideal vaccine must be free of adjuvant and have an excellent local tolerance.
Accordingly, the present invention seeks to address those problems evident in the traditional vaccines by utilizing a recent strain representative of the FCV population in an inactivated or recombinant vaccine (as opposed to a modified live vaccine) (Pedersen, 1995) that has improved local tolerance of the vaccine, and the FCV strain used in the vaccine must be broadly cross-protective—alternatively, the inclusion of several strains has been previously proposed (Baulch-Brown, 1997; Dawson, 1993; Knowles, 1990).